In general, the concentration of cholinesterase (hereinafter referred to as ChE) in serum is known to be decreased, for example, in a patient with liver disease, while it is known to be increased, for example, in a patient with kidney disease. Therefore, these disease can be diagnosed by determining the cholinesterase activity in serum of these patients and determination method which permits exact determination of cholinesterase activity in serum can be used for clinical examinations.
As method for determining cholinesterase activity in serum, there have heretofore been reported various methods using a synthetic substrate, and some of them have been made practicable for daily clinical examinations. Examples of the heretofore well-known determination methods include (a) gas analysis method, (b) pH meter method, (c) pH-indicator colorimetric method, (d) thiocholine color formation methods, (e) enzymatic method, (f) UV method, etc.
(a) The gas analysis method [R. Ammon: Pflugers Arch. Ges Physiol., 233, 487 (1933)]comprises using acetylcholine as a synthetic substrate, bringing acetic acid produced by the enzymatic action of ChE into contact with sodium hydrogen carbonate, and quantitatively determining carbon dioxide gas produced.
(b) The pH meter method [H. O. Michel: J. Lab. & Clin. Med., 34, 1564 (1949)], like the gas analysis method, comprises using acetylcholine as a synthetic substrate, and measuring a pH change due to acetic acid produced by the enzymatic action of ChE by means of pH meter.
(c) The pH-indicator colorimetric method, unlike the pH meter method, comprises using acetylcholine as a synthetic substrate, and measuring a pH change due to acetic acid produced by ChE in terms of the molecular absorbance of the indicator. As the indicator, there are used phenol red [Hiroshi Takahashi and Susumu Shibata, IGAKU-TO-SEIBUTSUGAKU (Medicine and Biology), 20, 96 (1959)], bromothymol blue [H. G. Biggs, et al., Amer. J. Clin. Path., 30, 181 (1958)], m-nitrophenol [Tadahide Sasaki, RINSHO-BYORI (chemical Pathology), 12, 555 (1964)], etc.
(d) The thiocholine method [P. Garry, J. Clin. Chem., 11, (2), 91 (1965)]uses acetylthiocholine, butylthiocholine or the like as a substrate. These substrate yields thiocholine by the enzymatic reaction of ChE, and then this thiocholine reacts with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to produce a yellow color. The thiocholine method comprises measuring this yellow color by means of a colorimeter.
(e) The enzymatic method comprises using benzoylcholine [Hirosaki Okabe et al, RINSHO-BYORI (clinical Pathology), 25, 751 (1977)], orthotoluoylcholine [Japanese Patent Application Kokai (Laid-Open) No. 138533/79]or the like as a substrate, converting chlorine produced by the enzymatic action of ChE into betaine by cholineoxidase, and subjecting 4-aminoantipyrine to oxidative condensation reaction with phenol or the like by thus produced hydrogen peroxide in the presence peroxidase to cause color production.
(f) The UV method includes two kinds of methods, and the one is a method of W. Kalow using benzoylcholine as a substrate [W. Kalow and K. Genet, Can. J. Biochem. & Physiol., 35, 339 (1975)], the another is a method using p-hydroxy-benzoylcholine [Japanese Patent Application Kokai (Laid-Open) Nos. 110198/82 and 129999/83]as a substrate. The former comprises monitoring a decrease in amount of the substrate caused by its hydrolysis due to the enzymatic action of ChE at a determination wave length of 240 nm. The latter comprises reacting p-hydroxy-benzoate hydroxylase with p-hydroxybenzoic acid produced by the enzymatic action of ChE, in the presence of the coenzyme NADPH, and monitoring, at a wave length of 340 nm, a decrease of absorbance upon the oxidation of NADPH into NADP by this reaction.
However, these determination methods involve various defects and problems, which are responsible for the inaccuracy of the resulting determined value. These method, for example, the gas analysis method (a) and the pH meter method (b), are disadvantageous in that their operations are troublesome and involves practical problems of inability to deal with many samples, and the like. The pH-indicator colorimetric method (c) comprises simple operations and can deal with many samples, but it should be pointed out that this method is disadvantageous, for example, in that the reaction time is long and in that during the reaction, the pH is not kept constant and the obtained values is not sufficiently reproducible at low and high values.
Any of the above-mentioned methods (a) to (c) use acetylcholine as a substrate, and in the case of such methods, the substrate itself also is disadvantageous because acetylcholine tends to undergo nonenzymatic hydrolysis and has no high substrate specificity.
The thiocholine method (d) is advantageous, for example, in that it is excellent in reactivity, has a high sensitivity, comprises simple operations, can deal with many samples, and make it possible to carry out the determination also by an initial velocity method. However, it is seriously affected by bilirubin in serum because of the yellow coloration and unavoidably affected by compounds having a thiol group such as glutathione. Furthermore, it is disadvantageous, for example, in that the substrate itself is instable. These disadvantages are responsible for errors of obtained values.
In the enzymatic method (e), since the coloration is red, there is not interference by bilirubin in serum, and many samples can be dealt with. However, since phenol or 4-aminoantipyrine used as a reagent for the color-producing system competitively inhibits ChE, the amount of these reagents used is greatly limited, so that sufficient color production is difficult. Also these enzymatic method utilizes hydrogen peroxide. In general, a determination method via hydrogen peroxide is unavoidably affected not only by bilirubine in serum, reducing substances such as ascorbic acid and the like but also by choline produced by decomposition of phospholipids or the like. In particular, the employment of benzoylcholine as a substrate involves various problems, for example, its non-enzymatic hydrolysis which causes troubles.
In the UV method (f), a method of W. Kalow, using benzoylcholine as substrate, measures directly a decrease in amount of the substrate. Consequently, the principle of determination of this method is simple and plain. However, this method is disadvantageous, for example, in that since the determination wave length is 240 nm, interference by serum components tends to occur, and in that since nonenzymatic hydrolysis of benzoylcholine tends to occur, the reaction can not be carried out in the optimum pH range of ChE. It is disadvantageous also, for example, in that since as the determination wave length there is used wave length at the slope of absorption spectrum of the substrate, resulting in a large deviation of absorption coefficient due to the deviation of wave length.
The UV method, using p-hydroxybenzoylcholine as a substrate, is an excellent method for determining ChE activity which makes it possible to carry out the reaction at a range of optimum pH, permits removal of the defects of the hydrogen peroxide coloration system, namely, removal of influence of bilirubin, reducing substance such as ascorbic acid and the like as well as interference by choline produced by decomposition of phospholipids, is free from the defects of the thiocholine method, and is applicable to an autoanalyzer capable of dealing with many samples. However, since NADPH, the coenzyme used, is an expensive reagent and is poor in stability, it is difficult to keep the coenzyme under a definite quality. Further, in this method, p-hydroxybenzoate hydroxylase, proto-catechuate 3,4-dioxygenase or the like is used as a reagent enzyme in the determination, therefore there are many factors which produce an error of the obtained value.
As described above, the conventional methods for determining the ChE activity involve various problems, and cause an error of the obtained value.